Mass spectrometric analysis of type 1 inositol 1,4,5-trisphosphate receptor ubiquitination.
Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric channels in endoplasmic reticulum membranes of mammalian cells and mediate IP(3)-induced calcium mobilization. In response to various extracellular stimuli that persistently elevate IP(3) levels, IP(3) receptors are also ubiquitinated and then degraded by the proteasome. Here, for endogenous type 1 IP(3) receptor (IP(3)R1) activated by endogenous signaling pathways and processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin conjugates. Our findings are (i) that at least 11 of the 167 lysines in IP(3)R1 can be ubiquitinated and that these are clustered in the regulatory domain and are found in surface regions, (ii) that at least approximately 40% of the IP(3)R1-associated ubiquitin is monoubiquitin, (iii) that both Lys(48) and Lys(63) linkages are abundant in attached ubiquitin chains, and (iv) that Lys(63) linkages accumulate most rapidly. Additionally, we find that not all IP(3)R1 subunits in a tetramer are ubiquitinated and that nontetrameric IP(3)R1 complexes form as degradation proceeds, suggesting that ubiquitinated subunits may be selectively extracted and degraded. Overall, these data show that endogenous IP(3)R1 is tagged with an array of ubiquitin conjugates at multiple sites and that both IP(3)R1 ubiquitination and degradation are highly complex processes.
Sliter
Danielle A
DA
Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Kubota
Kazuishi
K
Kirkpatrick
Donald S
DS
Alzayady
Kamil J
KJ
Gygi
Steven P
SP
Wojcikiewicz
Richard J H
RJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D013058:Mass Spectrometry; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
D053496:Inositol 1,4,5-Trisphosphate Receptors; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D002118:Calcium